Organic waste treatment

ABSTRACT

A method of treatment of sewage comprising adding at least one cell signalling chemical (CSC) to a sewage substrate, wherein the at least one CSC regulates activity of a microbial population in the sewage substrate. In particular, methods of reducing odor in sewage treatment systems, preventing corrosion in sewage treatment systems, enhancing microbial digestion of sewage, managing methane gas production in a sewage treatment system, enhancing digestion of sewage sludge, resuscitating dormant microbes or microbes that are in a starvation or stationary phase in a sewage substrate and controlling the bacteria responsible for oxidation or reduction of nitrogenous compounds in a sewage substrate, are provided.

The present invention relates to a method of modifying the activity ofmicrobial populations at the cellular level, in sewage, by the additionof at least one cell signalling chemical.

The decomposition of sewage is a significant problem on a global basis.The increasing trend towards urbanisation is a factor responsible forthe concentration of sewage in localised areas.

Sewage treatment plants are generally located away from populationcentres. With expanding urbanisation, the distance and the retentiontime of the sewage, in the sewerage catchment system before it reachesthe sewage treatment plant for processing is increased. Long retentiontimes and high temperatures create major problems within the seweragecatchment system as the sewage turns septic. Septic sewage is malodorousand difficult to process at sewage treatment plants (STP). This is inpart due to high sulfide levels inhibiting normal microbial activityassociated with the decomposition of sewage. This gives rise to effluentwaters that are not adequately digested and therefore comprise a highorganic fraction. Such effluent waters are potential health andenvironmental pollution hazards.

Septic sewage produces offensive odours such as the odour associatedwith hydrogen sulfide gas (rotten egg gas) and other malodorous gaseoussubstances. These odours, as well as being offensive, are toxic atspecific concentrations. High levels of dissolved sulfide's (for exampleHS or H₂S) within the sewage arriving at the STP must be oxidised tosulfates (for example SO₄ or H₂SO₄) increasing the energy requirementsfor oxidation. High levels of dissolved sulfides also inhibit sewagedigestion by aerobic and anaerobic methane-forming bacteria andtherefore may inhibit a significant component of the sewagedecomposition process, which may result in high sludge volumes.

In environments such as sewerage catchment systems, hydrogen sulfide gasis formed as a result of the microbial activity of sulfur reducingbacteria (equation 1 and 2).

Another group of bacteria may then convert the sulfides to sulfates,thereby producing sulfuric acid (equation 3).

The formation of sulfuric acid causes major corrosion problems (forexample equation 4) in sewerage catchment networks and sewage treatmentplants. Corrosion resulting from sulfuric acid is a major cause ofinfrastructure failure and degradation within these networks and plants.

Chemicals may be used to suppress both odour and corrosion in seweragesystems. Other chemicals may also be used to improve processing ofsewage. Examples of such chemicals include acid or alkali for pHcontrol, disinfectants, biocides, antibiotics, surfactants, deodorants,fragrances, chelating agents, oxidising compounds and oxygen gas.

Some chemical processes are reliant on a chemical reaction occurring toreduce odour, corrosion or microbial activity, while other chemicals maybe used to kill bacteria or specific groups of bacteria. Alternativelynutrients may be added to improve the environment of specific groups ofbacteria, so that they are then able to out-compete less desirablebacteria. Chemical buffering agents are used to improve sewage processwhile deodorants mask, absorb or react with odours.

In general, the suppression of odour and/or corrosion requires theaddition of large amounts of chemicals, many of which increase the saltlevels within the sewage effluent. Oxygen or oxidising compounds may beused to stimulate aerobic bacteria, facultative anaerobes and otheraerobic micro-organisms and thereby out-compete the sulfur reducingbacteria for the same food source.

There is a need for simple and effective methods of treating sewage toreduce or prevent the difficulties outlined above.

A number of naturally occurring chemicals are known to regulate thebehaviour of individual microbes or their communication with othermicrobes within a population. These chemicals are called Cell SignallingChemicals (CSC's). CSC's are diffusible signal molecules that act viamicrobial cell surfaces or intracellular receptors to modulate geneexpression. The CSC's are not bactericides or antibiotics and do notcause lysis of the bacteria cells. Similarly CSC's are not nutrientadditions that may be added to overcome a nutrient deficiency that mayregulate microbial numbers.

Specific concentrations of CSC's are known to produce a range ofresponses in prokaryotic cells, for example, autoregulation, stimulationof slow-growing or dormant microbes, quorum sensing, virulence,swarming, biofilm formation, increases or decreases in reproduction andincreases or decreases in metabolic activity. The fundamental differencebetween CSC's and other chemicals used in microbial control is that theCSC's manipulate microbial response via communication signals at thecellular level. These CSC's are responsible for switching on, orswitching off specific gene expression at specific signal strengths.CSC's do not kill bacteria nor do they provide essential nutrients toenhance or diminish the response of specific microbes. CSC's either upregulate (speed up) or down regulate (slow down), diminish or disruptthe communication signals between the bacteria. This down regulation mayeven force the bacteria to revert from a biofilm state to a planktonic(single cell or free floating phenotype) state. Conversely CSC's may upregulate (strengthen) the microbial communication signals. Thisincreased signal strength can be used to cause planktonic bacteria toincrease their reproduction and metabolic rate and thus exploit the foodresource available. The increased cell signal strength (CSC) may alsocause the planktonic bacteria to proliferate, attach to surfaces, formmicrocolonies, quorum sense and form mature biofilms.

The response of a specific species of bacteria to a specific CSC and itssignal strength is transient, if that specific signal and its strengthare not maintained. Both the signal strength and signal type areimportant in the maintenance of specific gene expression. If thespecific signal and signal strength are not maintained, the stimulatedgene will return to its non-stimulated state within varied periods oftime, generally ranging from minutes to hours. CSC's rapidly decomposein the environment and thus must be continually generated at specificsignal concentrations to elicit specific microbial responses.

Different microbes respond to different cell signalling chemicals atdifferent chemical cell signal strengths. For example, gram negativebacteria respond to N-acyl homoserine lactones, while gram positivebacteria respond to specific peptide pheromones, generally histidinekinases via a two component signal transduction system. The role ofCSC's has previously been observed in a range of laboratory experimentsand they have been used in the suppression of formation of biofilms inmarine environments. Such experiments and uses are described inKleerebezem M, Quandri L E N, Kuipers O P abd deVos W M. “Quorum sensingby peptide pheromones and two-component signal-transduction system inGram-positive bacteria” Molecular Microbiology, 1997, Vol 24 No. 5, pp895-904; de Nys R, Rice S, Manefield M, Kjelleberg S, et al. “Cross talkin bacterial extracellular signals”. Microbial Biosystems: NewFrontiers. Proceedings of the 8th International Symposium on MicrobialEcology. Bell C R, Brylinsky, Johnson-Green P (ed)., Atlantic CanadaSociety for Microbial Ecology, Halifax, Canada 1999; Lazazzera B A,Grossman A D “The ins and outs of peptide signalling” Trends Microbiol.,1998 July:6(7):288-94; Salmond G P, Bycroft B W, Stewart G S, Williams P“The bacterial ‘enigma’: cracking the code of cell to cellcommunication” Mol Microbiol., 1996 February;19(3):649; Buchenauer H.“Biological control of soil-bourne diseases by rhizobacteria”Zeitschrift-fuer-Pfanzenkrankheiten und Pflanzenschultz, July,1998;105(4)329-384; Kazlauskas R, Murphy P T, Quinn R J. Wells R J “Anew class of halogenated lactones from the red algae Delisea fimbriata(Bonnemaisoniaceae)” (1977) Tet. Lett. 1: 37-40; AntifoulingCompositions, Steinberg et al U.S. Pat. No. 6,060,046, 9 May 2000.

The present invention relates to the use of CSC's to manipulate,mediate, or regulate the communication signals between species orpopulations of bacteria in sewage, the sewerage catchment environment orthe STP. This enhances or disrupts the normal way in which bacteriacommunicate with each other. Using this technology, cell signals thatcause swarming, quorum sensing and the formation of biofilms may bedisrupted. This disruption may cause the bacteria to resume a planktonicstate and substantially down regulate their activity. Alternatively,other signals or signal strengths can be used to initiate or strengthenthe cell signals that cause resuscitation, swarming, increased metabolicactivity and reproduction rates, quorum sensing and the formation ofbiofilms. Advantageously, the CSC's may be used in very small quantitiesto elicit a desired response.

In one embodiment, the present invention provides a method of treatmentof sewage comprising:

-   -   adding at least one cell signalling chemical to a sewage        substrate, wherein the at least one cell signalling chemical        regulates activity in at least one microbial population in the        sewage substrate.

The activity of a microbial population may be modified or regulated bymanipulating single microbes within a population, by controlling thelevel of intra or inter cellular signalling chemicals. Alternatively,the activity of a microbial population may be modified or regulated bymanipulating a colony or population of microbes by controlling the levelof intercellular signalling chemicals within the medium. Specificmicrobial activities that may be manipulated by CSC's include cell tocell communication, quorum sensing, swarming, bacterial motility,symbiotic associations with multicellular organisms, cell metabolicrates, production of metabolic products, cell division and conjunction,cell resuscitation, formation of biofilm communities, entry into astationary or dormant phase, discrete and diverse metabolic processes inconcert with cell density, bioluminescence and the production ofantibiotics, surfactants and enzymes.

The term “cell signalling chemicals” (CSC's) refers to chemicals thatare capable of manipulating the behaviour of a specific microbialpopulation or populations through intra- or extra-cellular signals in orbetween prokaryotic cells. At specific CSC strengths, a specific speciesof microbe, responds to the signal at an intracellular level throughgene expression. These signals are generally used to assist a specificspecies of microbe to maximise its exploitation of a resource. DifferentCSC's may also mimic, block, inhibit or interfere with the communicationsignals between specific microbes or populations of microbes. Forexample, the cell signalling chemicals may act by binding to cellsurface receptors and inhibiting or blocking other CSC's therebymodifying communication between microbes in a population. This type ofCSC generally diminishes a specific microbial response or a population'sresponse and reduces their ability to exploit a resource. CSC's are alsoresponsible for regulating microbial activity by mediating cross talkbetween microbes. Cross talk appears important in mitigating starvationand stationary phase responses and in resuscitation of dormant orstationary bacteria.

As used herein, the term “up regulate” refers to the use of at least oneCSC at sufficient signal strength to cause a proliferation in microbialactivity of at least one species of microbe (an increase in either orboth the metabolic and reproduction rate of bacteria), the coordinationof individual microbial functioning resulting in the possible formationof an attachment layer on surfaces, the possible formation ofmicrocolonies, possible quorum sensing and the formation of maturebiofilms.

As used herein the term “down regulate” refers to the use of at leastone CSC at sufficient signal strength to cause a reduction in microbialactivity (a decrease in either or both the metabolic and reproductionrate of bacteria), the possible breakdown and dispersement of thebacteria forming the microbial attachment layer on surfaces, thepossible breakdown and dispersement of at least some species of bacteriaforming microcoloines, a reduction in quorum sensing by at least somespecies of bacteria and at least a reduction in some microbial speciesforming the biofilm. In many cases, down regulate will mean thedestruction of biofilm complex, with the individual bacteria or bacteriaspecies returning to a planktonic state while remaining viable andculturable. Down regulation may result from a decrease in signalstrength, from interference with the signals or existing signals, orfrom competition for the signal receptor sites on the bacteria.

The term “cross talk” as used herein refers to induced microbialreactions to a range of CSC signals and/or signal strengths. Differentpheromone and furanone signals and/or signal strengths are used tosignal or disrupt the signals between individual members of a bacterialpopulation (same species) or community (different species). Cross talkis important in strengthening or interfering with the communicationnetwork of competing bacteria. Cross talk enhances or diminishes the upor down regulation of bacteria and thus strengthens or diminishes amicrobial population or community's ability to exploit a resource. Crosstalk is also important in preventing bacteria from entering thestarvation or stationary phase and also in resuscitation.

It is particularly useful in the processing of sewage to modify theactivity of microbial populations to stimulate microbes that decomposeorganic waste and inhibit or down regulate the activity of microbes thatproduce toxic and malodorous gases such as sulfides, greenhouse gasessuch as methane or corrosive by-products such as sulfuric acid. It isalso useful to inhibit microbial populations that form biofilms thatsupport sulfur reducing bacteria in specific locations, such as in thesewerage catchment pipes.

Specific CSC's, controlling pheromones, for example, furanones andantimicrobial peptides, when applied to a sewerage catchment at specificconcentrations may be used to down regulate bacteria, swarming andprevent the subsequent formation of biofilms without the use ofintroduced products such as biocides that cause lyses of bacterialcells. Furanones and antimicrobial peptides may be used to cause thebacteria to disassociate, slough or remain in their planktonic (singlecell) state. This is of particular importance in the control of odourassociated with sulfur reducing and other odour forming bacteria. Intheir planktonic state, sulfur reducing bacteria will produce only aboutone thousandth of the level of sulfides that they are capable ofproducing in their biofilm state.

High levels of dissolved sulfides arriving at the head of works at theSTP, interfere with sewage processing. Minimizing the production ofsulfides in the sewerage catchment though the use of CSC's, means thatonly very low levels of sulfides will arrive at the STP and these willnot have a significant adverse impact on the sewage treatment process.Low levels of sulfides in the catchment will minimize the high levels ofcorrosion normally associated odorous sewage.

Specific CSC's, stimulating pheromones, for example, N-acyl homoserinelactone peptide and specific histidine protein kinase pheromones areparticularly useful in the processing of sewage. At specific dose rates,they are used among other things, to up regulate or stimulate themetabolic and reproduction rate of either or both aerobic and anaerobicbacteria and thereby enhance the decomposition of sewage. By controllingthe microbial populations, such factors as the level of aerobic andanaerobic decomposition and even the level of methane gas (a greenhousegas) produced can be controlled.

CSC's typically include bacterial pheromones, eukaryotic hormones anddiffusible communication molecules or their derivatives. CSC's may benaturally occurring or may be synthetic. Bacterial pheromones includecompounds such as N-acyl homoserine lactones (AHL's), pheromone peptidesincluding histidine protein kinases, N-acetylated, C-amidated D-aminoacid hexapeptides, D-amino acid peptides including D-tyrosine and/orD-isoleusine, cyclic dipeptides, hydrophobic tryamines, lipopeptidebiosurfactants, fatty acid derivatives, antimicrobial peptides andfuranones, such as halogenated, hydroxylated or alkyl furanones. CSC'smay also be Eukaryotic hormones including auxins, for example,indole-3-acetic acid, cytokinins or cytokines with cytokinin activitysuch as 6-(γγdimethylallylamino)purine, zeatin and 6-benzylamino-purine,ethylene gas, gibberellins and abscisic acid. Diffusible communicationmolecules are derived from plant, animal, algal or microbial sources andhave a cell signalling function or synthetic derivatives of thesecompounds. Other useful CSC compounds are Rhodomine 123 and3-hydroxypalmitic acid methyl ester.

Furanones that are useful in the present invention include4-acetoxy-2,5-dimethyl-3(2H)-furanone,4-hydroxy-5-methyl-3(2H)-furanone,4-hydroxy-2,5-dimethyl-3(2H)-furanone,4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone,4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone,4-hydroxy-5-methyl-3(2H)-furanone,4-methoxy-2,5-dimethyl-3(2H)-furanone,4-ethoxy-2,5-dimethyl-3(2H)-furanone,4-butyroxy-2,5-dimethyl-3(2H)-furanone,4-hydroxy-2,5-dimethyl-3(2H)-furanone,(S)-(+)-5-hydroxymethyl-2(5H)-furanone,(R)-dihydro-3-hydroxy-2(3H)-furanone,(S)-dihydro-3-hydroxy-2(3H)-furanone,(R)-dihydro-4-hydroxy-2(3H)-furanone,(R)-dihydro-5-(hydroxymethyl)-2(3H)-furanone,3-chloro-4-(bromochloromethyl)-5-hydroxy-2(5H)-furanone,3-chloro-4-(dibromomethyl)-5-hydroxy-2(5H)-furanone,3-bromo-4-(dibromomethyl)-5-hydroxy-2(5H) -furanone,3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and3-chloro-4-(dichloromethyl)-2(5H)-furanone. Acetyl homoserine lactones(AHL) include N-(3-oxohexanoyl)-L-homoserine lactone (OHHL),N-butanoyl-L-homoserine lactone (BHL), N-hexanoyl-L-homoserine lactone(HHL), butyryl homoserine lactone, hydroxybutyryl homoserine lactone,octanoyl homoserine lactone, 3-oxooctanoyl homoserine lactone,3R-hydroxy-7-cis-tetradecenoyl homoserine lactone and 3-oxododecanoylhomoserine lactone. Suitable histidine protein kinases include groupsHPK 1a, HPK 1ai, HPK 1b, HPK 1c, HPK 2a, HPK 2b, HPK 2c, HPK 3a, HPK 3b,HPK 3c, HPK 3d, HPK 3e, HPK 3f, HPK 3g, HPK 3h, HPK 3i, HPK 4, HPK 5,HPK 6, HPK 7, HPK 8, HPK 9, HPK 10 and HPK 11.

Pheromones are small molecules secreted by one individual prokaryote andreceived by a second individual of the same species in which they signala specific action. At specific signal strengths (chemicalconcentrations) the bacteria may swarm, quorum sense and form biofilms.Conversely, if the signal strength diminishes, the bacteria maydissociate, slough off and resume their planktonic state. Pheromones arepositive signals that up or down regulate the microbial response.

Furanones by contrast are signal blockers. That is, substances thatcompete with the pheromones for the same signal site, or block thepheromone signal in some other way. At specific signal strengths, afuranone can prevent at least one species of bacteria from forming acolony or it can cause the demise of colonies by interrupting the normalpheromone communication between the bacteria.

There are many pheromone and furanone signals and cross talk between thebacteria appears prevalent. Pheromones appear to be important interritory marking, with specific pheromone signals at specific strengthseliciting specific responses in the species of bacteria and thusallowing one species to out compete another species for an environmentalniche. Furanones, at low signal strengths, appear to have a role in themitigation of starvation and stationary phases, probably by interruptingthe pheromone signals that are telling the bacteria to enter astarvation or stationary phase.

Microbial populations that may be manipulated by the methods of theinvention include gram positive bacteria, gram negative bacteria,cyanobacteria, autotrophic bacteria (photosynthetic andchemoautotrophic), heterotrophic bacteria and nitrogen-fixing bacteria.The invention is also useful in manipulating populations of aerobes,facultative anaerobes and anaerobes; and is particularly useful inmanipulating populations of bacteria that produce malodorous gas,including hydrogen sulfide gas (produced by sulfur and sulfate reducingbacteria), and bacteria that convert sulfide to sulfate. The inventionis also useful in manipulating ammonia forming, nitrite forming, nitrateforming, denitrifying and methane forming bacterial populations.

In one embodiment of the invention, the activity of Gram negativebacteria is enhanced or inhibited by the addition of at least one CSC.At least one CSC is added to a sewage substrate, for example, anN-acylated homoserine lactone, at a specific dose rate to regulate,enhance, initiate and/or sustain specific functioning levels ofmicrobial activity including quorum sensing, swarming and biofilmproduction. Conversely, at least one other CSC, for example, ahalogenated furanone, may be added to a sewage substrate at a specificdose rate to down regulate functions such as swarming and biofilmattachment. Specific furanones at specific dose rates are used tointerfere with the interspecies communication and this causesdissemination of biofilms, sloughing and the maintenance of bacteria intheir planktonic state.

In another embodiment of the invention, the activity of Gram positivebacteria is enhanced or inhibited by the addition of at least one CSC.In this embodiment, the at least one specific CSC is a peptide pheromonethat activates the histidine protein kinases receptor of a two componentsignal transduction system. Specific dose rates of the at least one CSCare used to up or down regulate, enhance or diminish, initiate and/orsustain specific functioning levels of microbial activity, such asswarming, quorum sensing and biofilm production. Other CSC's, forexample antimicrobial peptides and furanones, that inhibit the histidineprotein kinase response regulator two component signal transductionpathway, are used at specific dose rates to disrupt the pheromonepeptide signals and thus down regulate the effect of swarming andbiofilm attachment. Such peptides include N-acetylated D-amino acidhexapeptides. Specific furanones at specific dose rates interfere withthe interspecies communication and thus cause the dissemination ofbiofilms, sloughing and the maintenance of bacteria in their planktonicstate.

In another aspect of the invention, the at least one CSC may impact onhistidine kinase protein receptors (signal receivers) or on the domainsof the response regulator proteins controlling phosphorylation orde-phosphorylation of the regulatory domains. The phosphorylatedresponse either stimulates or represses the transcription of specificgenes. However, the phosphorylated response is transient with theresponse regulator returning to the non-stimulated state within a variedperiod of time, for example, between a few seconds and a few hours. Tomaintain the desired state, it is important to maintain the at least oneCSC at sufficient signal strength to obtain the desired signal response.

In another aspect of the invention, the at least one CSC may downregulate the swrA gene and hence reduce the production of lipoproteinbiosurfactant required for swarming.

In another aspect of the invention the at least one CSC may be a cyclicdipeptide which may interfere with acylated homoserine lactones (AHLs)by competing with the AHLs for the bacteria's binding sites.

In yet another aspect, the at least one CSC may be a furanone applied ata non-growth inhibitory concentration, that will minimise or eliminatethe impact of stress resistance, senescence, or lack of culturabilityarising from carbon starvation. The at least one CSC may also contain asupernatant solution used in combination with the furanone to protectagainst the loss of culturability arising from carbon or other stresses.

In another aspect, at least one furanone (CSC) may be added to interferewith interspecies communication during swarming of mixed cultures. Thiswill reduce the production of serrawettin W2 that is essential for thesurface translocation of the differentiated swarm cells.

In another aspect, the at least one CSC can be used to control exoenzymeproduction and/or Harpin_(Ecc) production or control the genesresponsible for post-transcriptional regulation and therefore controlchanges in the phenotype or phenotype expression.

In yet another aspect of the invention, the at least one CSC is used tocontrol specific microbial gene expression. There is an array ofdifferent gene expressions, which may be enhanced or diminished throughthe use of specific CSC's and or signal strengths. These include but arenot limited to luminescence, the production of toxins, antibiotics,enzymes, polysaccharides and surfactants. Microbially produced enzymesand microbially produced surfactants, or the lack of these products,play an important role in sewage transport or digestion. Toxins andantibiotics are more territory markers and thus also play a role inspecific species microbial dominance. Thus by controlling geneexpression through the use of the at least one CSC, the microbialdominance can be changed as well as the products produced by thatdominance and hence the rate of sewage digestion.

In another aspect, the at least one CSC can be used to interfere withthe genes responsible for the formation and/or signal strength of3-oxydodecanoyl HSL and/or butryl HSL. The production of 3-oxydodecanoylHSL (a CSC) by at least some microbes is important in regulating thepolysaccharide production required to cause adhesion of biofilms to atleast some surfaces. Butryl HSL appears responsible in at least somemicrobes for polysaccharide production, which is required in theformation of biofilms. Hence 3-oxydodecanoyl HSL and/or butryl HSLappear to be important in the formation and/or attachment of biofilmsfor at least some microbes. Interfering with these signals or signalstrengths is important in minimising biofilm attachment and/or theformation of biofilms.

In another aspect, the at least one CSC may be a mimic of the bacterialpheromone or alternatively it may initiate the gene response initiatedby the bacterial pheromone. That is, the mimic will provideextracellular signals that will elicit the same responses as thebacterial pheromone when applied to a media at specific concentrations.

In another aspect, the at least one CSC may be a mimic of a bacterialfuranone. The mimic, when applied to sewage at specific concentrationswill provide the extracellular signals that will elicit the sameresponses as the bacterial furanone in the sewage.

In another aspect, the concentration of the at least one CSC (signalstrength) will be a critical factor in the activation of specificreceptor proteins that will initiate specific gene expression.

Sewage includes both carbonaceous and nitrogenous waste matter.Carbonaceous waste includes compounds containing carbon and hydrogenatoms and may include other atoms, such as oxygen, nitrogen, sulfur andphosphorus. Nitrogenous waste includes compounds containing nitrogenatoms and other atoms such as hydrogen, carbon and oxygen. Nitrogenouswaste includes urea, uric acid, ammonia, nitrates and nitrites. Sewageincludes populations of many different microbes, including aerobes,facultative anaerobes and anaerobes.

Sewage has a variable composition which depends on the substances beingdischarged into the sewerage catchment network. Furthermore, the flowrate of sewage in the sewerage catchment network is variable and thisdetermines the retention time of the sewage in the sewerage catchmentnetwork. For example, at night, the amount of sewage entering thesewerage catchment network is low and flow rates tend to be slow. In thedaytime, more sewage enters the sewerage catchment network, and itscomposition may vary as industrial waste products may be discharged intothe network as well as human waste. Furthermore, as greater amounts ofwaste are discharged into the sewerage catchment network, the flow rateof the sewage increases.

The dosage levels of CSC's required will depend on the volume, flowrate, Biochemical Oxygen Demand (BOD)/Chemical Oxygen Demand (COD)loading and bacterial composition of the sewage substrate. The bacterialcomposition of a sewage substrate may be determined using standard platecounts for bacterial species or types. The dosage level of CSC'srequired may be determined by assessing bacterial composition, volumeand flow rate, therefore, dosage levels may vary over a treatmentperiod, for example, a 24 hour period.

Typically the CSC's may be added to the sewage substrate at a dosagelevel of between 1 nanogram/liter or kilogram of sewage and 1 gram/literor kilogram of sewage. Furanones are effective at preventing biofilmformation or adhesion in sewerage catchment pipes in the range of 2.5μg/m² to 25 g/m².

One or more CSC may be added to a sewage substrate. A single CSC atspecific signal strength or concentration may modify the behaviour of asingle population (phenotype expression) of microbes in the sewagesubstrate or it may independently modify the behaviour (phenotypeexpression) of more than one population of microbes in the sewagesubstrate. Alternatively, a mixture of CSC's may be added to a sewagesubstrate that may together modify the behaviour of a single populationof microbes, or may independently modify the behaviour of more than onepopulation of microbes in the sewage substrate. A mixture of CSC's maybe added so that a second CSC supplements or complements the activity ofa first CSC. For example, by enhancing a response or preventing feedbackinhibition, which may otherwise desensitise a microbial cell's activityto the first CSC.

Alternatively, furanones and antimicrobial peptides (CSC's) may be addedto sewage in a catchment to prevent swarming and the colonization ofsurfaces by biofilms. These CSC's may be specific or general in theirapplication in maintaining bacteria in their planktonic state.Additional specific CSC's at specific concentrations (signal strengths)may be added to the sewage to mediate the starvation and stationaryphase responses. This cross talking between various bacterial signals(CSC's) and CSC signal strengths can be maintained during the transitphase of the sewage in the sewerage catchment pipes. On arrival of thesewage at the head of works at the STP, different signals or signalstrengths can be used to modify the microbial population in such a wayas to rapidly accelerate aerobic or anaerobic sewage digestion(decomposition), depending on the design requirements at the STP.

Advantageously, the at least one CSC may be added to the sewagesubstrate in a composition comprising a growth promoting media thatsupports or enhances the growth of desirable microbes in the sewagesubstrate leading to an increased rate of decomposition. Such growthpromoting media may contain carbohydrates, proteinaceous substances,amino acids, fats and oils, vitamins and minerals. However it must berecognised that it is the CSC's that are the primary regulators ofmicrobial activity. The growth media supplement simply removes the lackof growth limiting factors.

Compositions containing CSC's may also contain chemicals that react withtoxic or undesirable by-products of the decomposition processes therebyrendering them non-toxic or more desirable, to supplement or complementthe effects of the CSC's. For example, a compound may be added that willmediate the effects of fatty acids or other noxious compounds, or adjustthe pH and make the medium more or less suitable for a particular colonyor species of bacteria. Alternatively, the CSC may be added to thesewage substrate together with oxygen or an oxidising compound. The CSCstimulates the growth and metabolic rate of aerobic bacteria, while theoxygen supplied removes oxygen deficiency as a growth limiting factor.

At least one CSC or compositions containing at least one CSC may beadded to an isolated sewage substrate. Alternatively, they may be addedat specific points along a sewerage catchment network, at a sewagetreatment plant or may be added to the sewage effluent. The at least oneCSC or compositions containing at least one CSC may be added in a bolusor may be added continuously or at intervals by means of a simple dripfeed or a pumping system. The at least one CSC or compositionscontaining at least one CSC may be in a spray or drip form and may beadded to the sewage by spraying or dripping on to the surface of thesewage. At the point of adding the at least one CSC or compositionscontaining at least one CSC, there may be a means of mixing the CSCswith the substrate to assist dispersion of the at least one CSCthroughout the sewage substrate. There may also be a means of monitoringthe concentration of a CSC that is added to an organic waste substrateto ensure the desired concentration is maintained, additions of furtherCSC may be made if the concentration drops below a desired level, oraddition may be suspended if the concentration rises above a desiredlevel. A person skilled in the art will be able to determine anappropriate means and amounts of adding the CSCs or compositionscontaining them to a substrate.

The at least one CSC or composition containing at least one CSC may bein the form of liquids, solutions, powders, granules, pellets or agaseous form. Compositions containing at least one CSC may also containother suitable carriers or adjuvants. The compositions may also containcomponents such as dispersing agents, binders, wetting agents and othersurfactants, fillers or other components that complement or supplementthe activity of the CSC. Advantageously, the CSC's are generally addedto sewage back in the network catchment by means of peristaltic pumps,however, the CSC's may also be added in the form of slow-release pelletsor granules, allowing the CSC to be released into the substrate over aperiod of time. Alternatively they may be added to the system in agaseous form or dissolved into the liquid state.

In a preferred aspect of the invention there is provided a method oftreatment of a sewage substrate comprising:

-   -   adding at least one cell signalling chemical (CSC) to a sewage        substrate, wherein the at least one CSC enhances the activity of        aerobic, anaerobic or facultative anaerobic microbial        populations in the sewage substrate.

This aspect of the invention is particularly useful in up regulating themicrobial activity and hence increasing the rate of sewage decompositionin sewage treatment plants. It may also be used to promote theresuscitation of dormant, non-growing or slow-growing microbes that aidin the decomposition of sewage. These processes may be further enhancedby adding the cell signalling chemical in a composition containinggrowth promoting media: wherein the growth promoting media correctspossible nutrient imbalances that would otherwise impact by limiting theeffect of the CSC, on a specific microbial population or populations.

In yet another preferred aspect of the invention there is provided amethod of reducing microbial activity in the sewerage catchment networkcomprising:

-   -   adding at least one cell signalling chemical (CSC) to the        sewerage catchment network,    -   wherein adding the at least one CSC to a sewerage catchment        network down regulates aerobic, facultative anaerobic and        anaerobic microbial activity in a sewage substrate.

This aspect of the invention is particularly useful in reducing sewagedecomposition in the sewerage catchment network and for deliveringfresher sewage to the sewage treatment plant. Fresh sewage arriving atthe sewage treatment plant, allows for the optimisation of the aerobicdecomposition of sewage, in accordance with the engineering designcriteria for the sewage treatment plant.

In another preferred aspect of the invention there is provided a methodof reducing odour in sewage treatment systems comprising:

-   -   adding at least one cell signalling chemical (CSC) to a sewage        treatment system, wherein the at least one cell signalling        chemical inhibits (down regulates) the activity of the odour        producing microbes or enhances (up regulates) the activity of        other microbial populations in the sewage treatment system, so        that the other microbial populations out compete the odour        producing microbes for the same food source.

As used herein the term “sewage treatment system” refers to bothsewerage catchment network and sewage treatment plant.

This aspect of the invention is particularly useful in reducingmalodorous sulfide gases in decomposing sewage, particularly hydrogensulfide gas, which is produced by the activity of sulfur or sulfatereducing bacteria. Such gases may be produced in the sewerage catchmentnetwork or at the sewage treatment plant. The activity of othermicrobial populations may be enhanced thereby allowing those microbes toout-compete the sulfur reducing bacteria for the same food source.

The addition of at least one CSC to a sewerage catchment network, maydown regulate the behaviour of a sulfur reducing microbial population,causing them to dissociate from a biofilm or alternatively it may causethe dissociation of the biofilm resulting in the bacteria resuming theirplanktonic state, preventing swarming or quorum sensing, inhibitingtheir reproduction and/or reducing their metabolic rate. The sulfur orsulfate reducing bacteria in their planktonic form only produce about a1000^(th) of the amount of sulfide that they produce when in a biofilmform. Alternatively, the at least one CSC may up regulate the behaviourof desirable microbes by stimulating swarming, quorum sensing,increasing their reproduction and/or metabolic rate allowing them to outcompete the sulfur reducing bacteria for the same food source.

In yet another preferred aspect of the invention there is provided amethod of reducing or preventing corrosion in sewerage treatment systemscomprising:

-   -   adding at least one cell signalling chemical to a sewerage        treatment system, wherein the at least one cell signalling        chemical inhibits the activity of microbes that convert hydrogen        sulfide to hydrogen sulfate (sulfuric acid).

The CSC may suppress the production of dissolved sulfides and hydrogensulfide gas, thereby removing a food source for the microbes, oralternatively it may disrupt communication signals between sulfateproducing bacteria thus down regulating these bacteria and therebyreducing the amount of sulfate produced.

This aspect of the invention is particularly useful in preventingcorrosion in both the sewerage catchment network and the sewagetreatment plant.

In yet a further preferred aspect of the invention there is provided amethod of inhibiting the formation or maintenance of biofilms insewerage catchment networks comprising:

-   -   adding at least one cell signalling chemical to a sewerage        catchment network, wherein the at least one cell signalling        chemical inhibits swarming and quorum sensing in a population of        microbes.

As used herein the term “quorum sensing” refers to the communicationlevel, that is, signal type and signal strength, between bacteria thatdetermines if the bacteria remain in a planktonic state, swarm, coloniseor form biofilms. More specifically, on reaching a quorum, the phenotypeexpression of the bacteria changes as they change from a single cellularstate to a colony or multicellular state. Quorum sensing occurs whennumerous bacteria communicate intercellularly to regulate thetranscription of multiple target genes in concert with cell density.Under natural conditions quorum sensing is mediated through theproduction of one or more pheromones and signal strength is a criticalfactor in determining if a quorum exists and changes planktonicbacterial behaviour through mediated gene expression to that of acolony.

As used herein the term “biofilm” refers to the slimes that are formed,generally on the surfaces of objects when colonised by bacteria. Ofparticular interest are:

-   -   the biofilms that cause bulking in sludges    -   the biofilms that form on the walls of sewerage catchment pipes        and the biofilms that form on sediment depositions in the        sewerage catchment pipes.

The biofilm complex that forms filamentous bulking in sludges, reducesthe activated sludge processing ability and settling ability. Thisaspect of the invention is useful in reducing/preventing filamentousbulking of sludges and improving sewage processing and sludge digestion.The CSC may be used to control the formation and/or maintenance ofpopulations of filimentous bacteria responsible for bulking in sludges.

CSC's may be added to a sewage substrate to prevent biofilm productionand/or its adhesion to surfaces as the CSC disrupts the bacteria'sability to form an adhesion layer to the substrate and form biofilms orthe CSC prevents the formation of polysaccharides necessary in theformation of biofilms. The use of furanones to block N-acyl homoserinelactones such as 3-oxo-decanoyl homoserine lactone and butyrylhomoserine lactone or mixtures thereof is a particularly useful aspectof the invention as it prevents the adhesion and/or polysaccharideproduction necessary for the formation of biofilms.

Biofilms that form on surfaces may contain sulfur reducing bacteria aspart of the biofilm complex structure. The use of CSC's to removebiofilms or the sulfur reducing bacteria from biofilms is an importantaspect of the invention, as it prevents the formation of the malodorousgas, hydrogen sulfide, and other malodorous gases in the seweragecatchment network.

When forming biofilms, microbes use a sophisticated system ofintracellular communication signals (CSC). Disrupting the intracellularcommunication signals (CSC) prevents a biofilm forming or alternativelydisperses a biofilm as the communication signal or signal strength isnot maintained. The prevention or dispersal of specific biofilmscontaining anaerobic sulfur reducing bacteria in organic waste seweragecatchments, assists in the control of sulfur reducing microbes andtherefore controls odour in the sewerage catchment and at the sewagetreatment plant. Reduced levels of dissolved sulfides in the influentsewage arriving at the sewage treatment plant, also enhances processingof the sewage at the sewage treatment plant.

In yet another preferred aspect of the invention there is provided amethod of enhancing microbial digestion of sewage at a sewage treatmentplant comprising:

-   -   adding at least one cell signalling chemical (CSC) to a sewage        substrate at the sewage treatment plant or in the sewerage        catchment network,        wherein the at least one cell signalling chemical (CSC) enhances        the activity of aerobic and facultative anaerobic bacteria.

This aspect of the invention is particularly useful in treating sewageas it arrives at the sewage treatment plant. Advantageously, an increasein aerobic and facultative anaerobic activity, reproduction and/ormetabolic rates aids microbial digestion of the sewage which improvesthe sewage effluent quality and reduces sludge volumes. Particularlyuseful in this aspect of the invention are AHL's, pheromone peptides,N-acylated, C-amidated D-amino acid hexapeptides, D-amino acidscomprising D-isoleucine and/or D-tyrosine, cyclic dipeptides,hydrophobic tryamines, lipopeptide biosurfactants, fatty acidderivatives, antimicrobial peptides and furanones. Especially preferredCSC's are AHL's.

In yet another preferred aspect of the invention there is provided amethod of managing methane gas production at a sewage treatment plantcomprising:

-   -   adding at least one cell signalling chemical (CSC) to a sewage        substrate in the sewerage catchment network or at the sewage        treatment plant,        wherein the at least one cell signalling chemical (CSC) enhances        or inhibits the activity of anaerobic, methane forming bacteria.

This aspect of the invention is particularly useful for managing methanegas production. This is desirable as methane gas is a “greenhouse gas”.Better management of methane gas production, may facilitate itscollection and its conversion to carbon dioxide and water, throughcombustion of this gas.

In yet another preferred aspect of the invention there is provided amethod of controlling the bacteria responsible for the oxidation orreduction of nitrogenous compounds in a sewage substrate comprising:

-   -   adding at least one cell signalling chemical (CSC) to the sewage        substrate, wherein the at least one CSC regulates the activity        of ammonia producing bacteria, nitrite producing bacteria,        nitrate producing bacteria or denitrifying bacteria.

Specific CSC's or combinations of CSC's and/or specific CSC signalstrengths can be used to up or down regulate the bacteria responsiblefor the ammonification, nitrification and denitrification of sewage.This aspect of the invention is particularly useful in controlling arange of environmental pollutants, both airborne and waterborne. N-acylhomoserine lactones such as 3-oxo-decanoyl homoserine lactone andbutyryl homoserine lactone or mixtures thereof are particularly usefulin up regulation, while halogenated furanones, hydroxylated furanonesand alkyl furanones are particularly useful in the down regulation ofthis aspect of the invention.

In yet another preferred aspect of the invention there is provided amethod of enhancing digestion of sewage sludge comprising:

-   -   adding at least one cell signalling chemical (CSC) to a sewage        substrate,        wherein the at least one cell signalling chemical enhances the        activity of aerobic or anaerobic bacteria.

This aspect of the invention is particularly useful in increasing therate of aerobic or anaerobic non-sulfur reducing bacterial digestion ofsewage sludge thereby reducing sludge volumes and odour.

In yet another aspect of the invention there is provided a method ofresuscitating dormant microbes, or microbes that are in a starvation orstationary phase in a sewage substrate comprising:

-   -   adding at least one cell signalling chemical (CSC) to a sewage        substrate,        wherein the at least one cell signalling chemical (CSC)        stimulates activity in the dormant, starved or stationary        microbes.

This aspect of the invention allows the resuscitation of desirablemicrobes that are present in the sewage but are not active. Sewage isoften a toxic and hazardous environment for microbes. Numerous toxichousehold and industrial chemicals are poured down sewers. These toxicchemicals often have adverse impacts on microbes, causing them to sporeform, or simply down regulate to a dormant or stationary phases. Beingable to up regulate and resuscitate beneficial microbes using CSC's isoften important in improving sewage process.

Long transport distances of sewage can cause carbon starvation in thesewage, again causing bacteria to down regulate to a dormant orstationary phase, or spore form. The use of CSC's can be important inmitigating carbon starvation by down regulating the bacteria at onestage and then using CSC's to up regulate and/or resuscitate bacteria atanother stage in the sewerage catchment, or at the sewage treatmentplant. This aspect of the invention is important in mitigating theeffects of carbon starvation stress and poor sewage processing.Especially preferred CSC's useful in this aspect of the invention arefuranones.

Throughout this specification, unless the context requires otherwise,the word “comprise”, and variations such as “comprises” and“comprising”, will be understood to imply the inclusion of a statedinteger or step or group of integers or steps but not the exclusion ofany other integer or step or group of integers or steps.

DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically represents the response of aerobic microbes in asewage sample to a biosol 1 mix of CSC's, where bacterial numbers aremeasured by standard plate counts.

FIG. 2 graphically represents the response of anaerobic microbes in asewage sample to a biosol 1 mix of CSC's, where bacterial numbers aremeasured by standard plate counts.

FIG. 3 graphically represents the response of sulfur reducing microbesin a sewage sample to a biosol 2 mix of CSC's, where bacterial numbersare measured by standard plate counts.

FIG. 4 graphically represents the response of aerobic microbes in asewage sample to different CSC's set out in Table 1, where samples havebeen aerated.

FIG. 5 graphically represents the response of anaerobic microbes in asewage sample to different CSC's set out in Table 1, where samples havebeen aerated.

FIG. 6 graphically represents the response of sulfur reducing microbesin a sewage sample to different CSC's set out in Table 1, where sampleshave been aerated.

FIG. 7 graphically represents the response of aerobic microbes in asewage sample to different CSC's set out in Table 1, where samples havenot been aerated.

FIG. 8 graphically represents the response of anaerobic microbes in asewage sample to different CSC's set out in Table 1, where samples havenot been aerated.

FIG. 9 graphically represents the response of sulfur reducing microbesin a sewage sample to different CSC's set out in Table 1, where sampleshave not been aerated.

FIG. 10 graphically represents the average amount of dissolved sulfidesin a sewage sample over a period of time during which sloughing andreduction of a biofilm is observed.

EXAMPLES Example 1

Methodology for testing the effectiveness of various CSC's dose rates.

The methodology is reliant on well defined NATA certified procedures(Australian, British or American Standards) for standard plate(heterotrophic) microbial counts. The plate counts undertaken are:

-   -   Aerobic,    -   Anaerobic including facultative anaerobes and    -   Sulfur reducing bacteria.

Sampling procedures are reliant on taking predefined sewage sample. Thesewage sample is then broken down into sub-samples of 800 ml. One ofthese sub-samples remains as the control, while the CSC's being tested,are added in specific amounts (generally nanograms to milligrams/liter)to other identical sub-samples.

Each of the 800 ml sub-sample of sewage, is then shaken to thoroughly toensure that the sewage is mixed with the added CSC's. The controlsub-sample is also shaken for an identical time and in an identicalmanner, to ensure consistency between the sub-samples.

Each sub-sampled is then decanted into 4 identical 200 ml samples calledA, B, C & D.

The A samples from each replication is then subjected to the NATAcertified procedures for standard plate counts.

If testing for aerobic microbial response, the B, C and D samples areaerated by means of an air pump injecting a small but steady stream ofair into the base of the sample and allowing the air to bubble upthrough the sewage mix for two minutes every four hours.

If testing for an anaerobic response, each 200 ml B, C & D sample istightly capped with an airtight cap to prevent further air entering thesample.

After 24 hours the B samples from each replication are then subjected tothe NATA certified procedures for standard plate counts in an identicalmanner to the A samples.

After 48 hours the C samples from each replication are then subjected tothe NATA certified procedures for standard plate counts in an identicalmanner to the A samples.

After 72 hours the D samples from each replication are then subjected tothe NATA certified procedures for standard plate counts in an identicalmanner to the A samples.

Example 2

Using the procedure outlined in Example 1, a mix of CSCs (Biosol 1 or 2)was added to a sewage sample. The compositions termed Biosol 1 andBiosol 2 give an indication of what can be achieved with a relativelyfresh sewage sample taken from the head of works at a small sewagetreatment plant. The results are shown in FIGS. 1 to 3.

The composition of Biosol 1 is:

-   -   0.05 mg/L N-(3-oxohexanoyl)-L-homoserine lactone    -   0.05 mg/L 3-oxydodecanoyl homoserine lactone    -   0.005 mg/L N-butanoyl-L-homoserine lactone    -   0.01 mg/L Zeatin    -   0.08 mg/L 6(γγ-dimethyallylamino) purine    -   0.08 mg/L 6-benzylamino-purine    -   0.1 mg/L 3-hydroxypalmitic acid methyl ester    -   0.1 g/L extract from Delisea pulchra (a known source of        furanones) the contents of half of one 1000 mg multivitamin        capsule    -   1 g of yeast extract    -   0.1 g seaweed powder (for minerals) (Durvillea potatorun)

The mixture was made up to one liter using deionised water. This mix wasadded at a rate of 4 mg/L sewage.

The composition of Biosol 2 is:

-   -   0.005 mg Rhodomine    -   2 g/L extract from Delisea pulchra (a known source of furanones)    -   0.005 mg/L N-butanoyl-L-homoserine lactone the contents of half        of one 1000 mg multivitamin capsule    -   1 g yeast extract    -   0.1 g seaweed powder (for minerals) (Durvillea potatorun).

The mixture was made up to one liter using deionised water. This mix wasadded at a rate of 4 mg/L sewage.

FIG. 1 shows that the mix of CSCs (Biosol 1) initially suppressedaerobic microbial activity, prior to a substantial increase in microbialnumbers, as indicated by the number of colony forming units. The controlshowed an initial rapid response in microbial activity, but then failedto respond to the available food source.

FIG. 2 shows that the mix of CSCs (Biosol 1) resulted in a rapidincrease in anaerobic microbial populations in raw sewage.

FIG. 3 shows that the mix of CSC's in Biosol 2 causes the reduction insulfur reducing bacteria colony forming units. Although the controlinitially showed a reduction in sulfur reducing bacteria, the sulfurreducing bacteria increased in numbers after 28 hours of incubation.

Example 3

Using the procedure outlined in Example 1, specific CSC's were added tosewage samples in vitro at specific dose rates. Each sample containing adifferent CSC as outlined in Table I below and each sample was dividedinto three sub-samples. The first sub-samples were plated within 4 hoursfor analysis by standard plate counts for aerobic, anaerobic and sulfurreducing bacteria The second and third sub-samples were aerated every 4hours and analysed as for sub-sample 1 at 24 hours and 76 hoursrespectively. The population density of aerobic, anaerobic and sulfurreducing bacteria were determined by the number of colony forming units,counted from each sewage sample analysed.

The results are shown in FIGS. 4-6. The results show that differentCSC's applied to sewage samples at different dose rates could markedlychange the microbial population growth rates when compared with thecontrol.

TABLE I Group CSC added Amount added 1 no CSC-Control — 2 Acylhomoserine lactone 0.5 mg/L 3 Zeatin 0.1 mg/L 6-(γγ-dimethylallylamino)purine 0.1 mg/L 6-benzylamino-purine 0.1 mg/L Indole-3-Acetic Acid 0.1mg/L made up to 1 L 4 kinetin   1 mg/L 5 Rhodamine 123  50 ng/L

Example 4

The experiment of Example 3 was repeated but the samples were notaerated. The results are shown in FIGS. 7 to 9.

Example 5

CSC's in the form of Biosol 2 was added at a rate of 4 ppm to acomponent of a sewage catchment. Hydrogen sulfide gas readings werehalved within 24 hours and the level of dissolved sulfides in the sewagedecreased by 48% during the same time.

Example 6

Biosol 1 was added at a rate of 4 ppm to the catchment of a small sewagetreatment plant handling a septic high organic load. It allowed thedissolved oxygen levels in the aerobic chamber to increase from lessthan 1 ppm to around 7 ppm. It reduced sludge volumes by 52% andsuspended solids in the effluent by 80%.

The results of Examples 5 and 6 are in line with the expectations fromthe in vitro experiments.

Example 7

A mix of halogenated furanones (1 g/L) in distilled water were addedusing a peristaltic pump at a rate of approximately 0.5 mg/liter tosewage flowing in a small gravity sewage pipe containing an establishedbiofilm matrix. After one month a noticeable reduction in the biofilm onthe pipe walls and bare patches of pipes were evident. The mix ofhalogenated furanones appeared to cause the sloughing off of biofilmsfrom the sewage pipe walls and affected the formation of biofilms.

Example 0.8

An equal mix of halogenated furanones, hydroxylated furanones and alkylfuranones (1 g/L) and AHL's (1 g/L) were mixed in distilled water andwere added using a peristaltic pump at a rate of approximately 4 ml ofmixture per liter of sewage flowing in a small gravity sewage pipecontaining an established biofilm matrix. After one month their was anoticeable reduction in the biofilm on the pipe walls and bare patchesof pipes were also evident. The furanone mix appeared to disrupt the AHLsignals and cause the sloughing off of biofilms from the sewage pipewalls and affect the formation of biofilms.

Example 9

An extract from Delisea pulchra (collected from Cape Banks NSW chilledand freeze dried within 24 hrs) was prepared by vitamising and usingdichloromethane to extract the CSC's from the Delisea pulchra. Theextract and crude fibre were reduced in vacuo prior. 10 g of fibre andextract per liter of water were then vitamised for 20 minutes withsufficient ascorbic acid to lower the mix to pH 3.5. The liquid wasallowed to settle for 2 hrs and the supernatant collected for use. Thisextract which is a known source of furanones was added to a sewagepipeline at an approximate rate of 4 ml/l of sewage flowing in the pipeover 4 weeks. After 4 weeks the extract appeared to cause the sloughingoff of biofilms from the sewage pipes and affect biofilm formation.

Examples 7, 8 & 9 were also used to measure the impact on dissolvedsulfide production. Initially there was a rise in dissolved sulfidesfollowed by a fall in the levels of dissolved sulfides in the sewage.The initial rise in dissolved sulfides was attributed to high levels ofdissolved sulfides in the sloughing biofilm. The decrease in the rate ofdissolved sulfide production was attributed to a reduction in thebiofilm and a change in the microbial matrix of the biofilm. See Table 2and FIG. 10.

TABLE 2 Average results Expt 7, 8 & 9 Day ppm 1 18 2 21 3 17 4 22 5 23 619 7 14 8 16 9 11 10 13

Example 10

A mix of halogenated furanones (1 g/L) was added to each liter of theDelicia pulchra extract. This mix was added at a rate of approximately 4mg/liter by a pump station, to the pumped sewage flowing in a smallpressure sewerage main. This pressure main contained an establishedbiofilm matrix. The sewage as it exited the pressure main was anaerobicand became highly odorous as it moved into the gravity main. The gaseswere believed to be mainly composed of hydrogen sulfide but othermalodorous gases such as mercaptans, indoles and skatoles were present.Prior to adding the mix above, hydrogen sulfide gas levels averagedaround 180 ppm at the end of the pressure main. One week after addingthe above mix, the hydrogen sulfide gas levels had decreased to anaverage of 47 ppm. One month after adding the mix the hydrogen sulfidelevels had reduced to average 4 ppm. Although not specifically tested,there was a noticeable absence of mercaptan, indole and skatole typeodours.

An “OdaLog” (0-1000 ppm H₂S) gas loggers, was suspended in a manhole atthe end of the pressure main, over 24 hours to measure H₂S gas levels.The average reduction in the H₂S gas level was calculated from thesereadings.

Example 11

A mix of halogenated furanones (1 g/L) was added to each liter of theDelicia pulchra extract. This mix was added at a rate of approximately 4mg/liter, to sewage flowing in a sewerage pipe containing an establishedbiofilm matrix. The pipe contained sewage that was septic, but this wasaerated as the sewage tumbled into a wet pump well. The nitrous oxidereleased into the pump well chamber at the end of the pipe was measuredwith a NO₂ gas data logger from 5.40 am to 9.00 am for 4 days. While thelevel of NO₂ gas varied as expected, the average reduction was about 70%with the addition of the above mix to the sewage sampled.

TABLE 3 No added Mixture Added Mixture % Change in NO₂ Time AM NO₂ppm(control) NO₂ ppm produced 5.40 19.69 7.50 38% 6.00 11.79 3.75 32%6.20 17.50 10.63 61% 6.40 32.77 16.88 51% 7.00 30.89 17.77 58% 7.2014.29 9.82 69% 7.40 11.96 0.00 100%  8.00 16.96 0.89 95% 8.20 20.45 1.7991% 8.40 25.89 0.00 100%  9.00 17.41 0.00 100% 

Example 12

The same mix as used in Example 11 above was sprayed on and around anodorous men urinal. The odour was eliminated within an hour. Thisindicated that urea was not being converted to ammonia, the major odoursource around stale urinals.

1. A method of treatment of sewage comprising: assessing at least one ofvolume, flow rate, Biochemical Oxygen Demand/Chemical Oxygen Demandloading, and bacterial composition of said sewage to determine a dosageof at least one cell signaling chemical (CSC); and adding the dosage ofthe at least one cell signalling chemical to said sewage, wherein the atleast one CSC regulates activity in at least one microbial population insaid sewage by modulating gene expression within a microbial cell ormodulating communication signals between microbial cells or populationsof microbial cells, wherein the microbial population includes aerobicbacteria, facultative anaerobic bacteria, anaerobic bacteria, orcombinations thereof, and wherein the at least one CSC does not kill thebacteria in the microbial population.
 2. The method according to claim 1wherein the at least one CSC is a bacterial pheromone, a eukaryotichormone or a diffusible communication molecule.
 3. The method accordingto claim 2 wherein the bacterial pheromones are selected from the groupconsisting of N-acyl homoserine lactones (AHLs), pheromone peptides,N-acetylated, C-amidated D-amino acid hexapeptides, D-amino acidpeptides comprising D-isoleucine and/or D-tyrosine, cyclic dipeptides,hydrophobic tryamines, lipopeptide biosurfactants, fatty acidderivatives, antimicrobial peptides and furanones.
 4. The methodaccording to claim 1 wherein the eukaryotic hormones are selected fromthe group consisting of auxins, cytokinins, cytokines, ethylene gas,gibberellins and abscisic acid.
 5. The method according to claim 1wherein the at least one CSC is Rhodomine 123 and/or 3-hydroxypalmiticacid methyl ester.
 6. The method according to claim 1 wherein the sewageis in a sewerage catchment network or a sewage treatment plant.
 7. Themethod according to claim 1 wherein the activity that is regulated isselected from the group consisting of cell to cell communication, quorumsensing, swarming, bacterial motility, symbiotic associations withmulticellular organisms, cell metabolic rates, production of metabolicproducts, cell division and conjunction, cell resuscitation, formationof biofilm communities, entry into a stationary or dormant phase,discrete and diverse metabolic processes in concert with cell density,antibiotic production and bioluminescence.
 8. The method according toclaim 1 wherein the microbial population is selected from the groupconsisting of Gram positive bacteria, Gram negative bacteria,cyanobacteria, autotrophic bacteria, heterotrophic bacteria andnitrogen-fixing bacteria.
 9. The method according to claim 1 wherein themicrobial population is selected from the group consisting of sulfurreducing bacteria, sulfate reducing bacteria, bacteria that convertsulfide to sulfate, ammonia producing bacteria, nitrite producingbacteria, nitrate producing bacteria and methane producing bacteria. 10.The method according to claim 1 wherein the activity of a microbialpopulation is up regulated, initiated or sustained by the addition ofthe at least one CSC.
 11. The method according to claim 10 wherein theat least one CSC is selected from the group selected from N-acylhomoserine lactones, histidine protein kinase pheromones, N-acetylated,C-amidated D-amino acid hexapeptides, D-amino acid peptides comprisingD-isoleucine and/or D-tyrosine, cyclic dipeptides, hydrophobictryamines, lipopeptide biosurfactants, fatty acid derivatives,antimicrobial peptides furanones, Rhodomine 123 and 3-hydroxypalmiticacid methyl ester.
 12. The method according to claim 11 wherein the CSCis an N-acyl homoserine lactone.
 13. The method according to claim 1,wherein the activity of a microbial population is down regulated by theaddition or reduction in signal strength of the at least one CSC. 14.The method according to claim 13 wherein the at least one CSC isselected from the group selected from N-acyl homoserine lactones,histidine protein kinase pheromones, N-acetylated, C-amidated D-aminoacid hexapeptides, D-amino acid peptides comprising D-isoleucine and/orD-tyrosine, cyclic dipeptides, hydrophobic tryamines, lipopeptidebiosurfactants, fatty acid derivatives, antimicrobial peptides,halogenated, hydroxylated or alkyl furanones, furanones, Rhodomine 123and 3-hydroxypalmitic acid methyl ester.
 15. The method according toclaim 14 wherein the CSC is a halogenated, hydroxylated or alkylfuranone.
 16. The method according to claim 1 wherein the at least oneCSC is added to regulate microbial gene expression.
 17. The methodaccording to claim 16 wherein the microbial gene expression is forcontrol of luminescence or the production of toxins, antibiotics,enzymes, polysaccharides and surfactants.
 18. The method according toclaim 1 wherein the activity of at least one of anaerobes, facultativeanaerobes, and aerobic microbial populations are up or down regulatedand the at least one CSC is selected from the group consisting ofN-(3-oxohexanoyl)-L-homoserine lactone, 3-oxydodecanoyl homoserinelactone, N-butanoyl-L-homoserine lactone, Zeatin,6-(-(γγ-dimethylallylamino)purine, 6-benzylamino-purine,3-hydroxypalmitic acid methyl ester, extracts of Delisea pulchra,kinetin, indole-3-acetic acid, Rhodomine 123 and mixtures thereof. 19.The method according to claim 1 wherein the activity of sulfur reducingbacteria is down regulated and the at least one CSC is selected from thegroup consisting of N-(3-oxohexanoyl)-L-homoserine lactone,3-oxydodecanoyl homoserine lactone, N-butanoyl-L-homoserine lactone,Zeatin, 6-(-(γγ-dimethylallylamino)purine, 6-benzylamino-purine,3-hydroxypalmitic acid methyl ester, extracts of Delisea pulchra,kinetin, indole-3-acetic acid, Rhodomine 123, 3-oxo-decanoyl homoserinelactone, butyryl homoserine lactone, halogenated furanones, hydroxylatedfuranones, alkyl furanones, and mixtures thereof.
 20. The methodaccording to claim 1 wherein the at least one CSC is added at intervalsas a bolus or is added continuously.
 21. The method according to claim 1wherein the at least one CSC is added to the sewage in a seweragecatchment network.
 22. The method according to claim 1 wherein the atleast one CSC is added to the sewage in a sewage treatment plant.
 23. Amethod of reducing odour in a sewage treatment system which includes asewerage catchment network and a sewage treatment plant comprising:assessing at least one of volume, flow rate, Biochemical OxygenDemand/Chemical Oxygen Demand loading, and bacterial composition ofsewage in said sewage treatment system to determine a dosage of at leastone cell signaling chemical (CSC); and adding the dosage of the at leastone cell signalling chemical to said sewage, wherein the at least oneCSC inhibits the activity of odour producing microbes or enhances theactivity of other microbial populations in the sewage treatment systemby modulating gene expression within a microbial cell or modulatingcommunication signals between microbial cells or populations ofmicrobial cells so that the other microbial populations out compete theodour forming microbes for the same food source, and wherein the atleast one CSC does not kill said odour forming microbes or said othermicrobial populations.
 24. The method according to claim 23 wherein theodour producing microbes are sulfur or sulfate reducing bacteria orammonia producing bacteria.
 25. The method according to claim 23 whereinthe at least one CSC is selected from the group consisting of bacterialpheromones, eukaryotic hormones and diffusible communication molecules.26. The method according to claim 23, wherein the at least one CSC isselected from the group consisting of N-(3-oxohexanoyl)-L-homoserinelactone, 3-oxydodecanoyl homoserine lactone, N-butanoyl-L-homoserinelactone, Zeatin, 6-(-(γγ-dimethylallylamino)purine,6-benzylamino-purine, 3-hydroxypalmitic acid methyl ester, extracts ofDelisea pulchra, kinetin, indole-3-acetic acid, Rhodomine 123,3-oxo-decanoyl homoserine lactone, butyryl homoserine lactone,halogenated furanones, hydroxylated furanones, alkyl furanones, andmixtures thereof.
 27. A method of reducing or preventing corrosion in asewage treatment system which includes a sewerage catchment network anda sewage treatment plant comprising: assessing at least one of volume,flow rate, Biochemical Oxygen Demand/Chemical Oxygen Demand loading, andbacterial composition of sewage in said sewage treatment system todetermine a dosage of at least one cell signaling chemical (CSC); andadding the dosage of the at least one cell signalling chemical to saidsewage, wherein the at least one CSC inhibits the activity of microbesthat convert sulfide to sulfate by modulating gene expression within amicrobial cell or modulating communication signals between microbialcells or populations of microbial cells, and wherein the at least oneCSC does not kill said microbes.
 28. A method of inhibiting theformation or maintenance of biofilms in sewerage catchment networks orsewage treatment plants comprising: assessing at least one of volume,flow rate, Biochemical Oxygen Demand/Chemical Oxygen Demand loading, andbacterial composition of sewage in said sewerage catchment network orsaid sewage treatment plant to determine a dosage of at least one cellsignaling chemical (CSC); and adding the dosage of the at least one cellsignalling chemical to said sewage, wherein the at least one CSCinhibits swarming, quorum sensing or biofilm attachment in a populationof microbes by modulating gene expression within a microbial cell ormodulating communication signals between microbial cells or populationsof microbial cells, and wherein the at least one CSC does not kill saidmicrobes.
 29. The method according to claim 28 wherein the at least oneCSC is selected from the group consisting of bacterial pheromones,eukaryotic hormones and diffusible communication molecules.
 30. Themethod according to claim 28 wherein the at least one CSC is selectedfrom the group consisting of N-acyl homoserine lactones (AHLs),pheromone peptides, N-acetylated, C-amidated D-amino acid hexapeptides,D-amino acid peptides comprising D-isoleucine and/or D-tyrosine, cyclicdipeptides, hydrophobic tryamines, lipopeptide biosurfactants, fattyacid derivatives, antimicrobial peptides and furanones.
 31. The methodaccording to claim 28 wherein the at least one CSC is selected fromhalogenated, hydroxylated or alkyl furanones.
 32. The method accordingto claim 28 wherein the at least one CSC is selected from 3-oxo-decanoylhomoserine lactone, butyryl homoserine lactone, extracts of Deliseapulchra and mixtures thereof.
 33. The method according to claim 28wherein the CSC down regulates production of lipoprotein biosurfactantin a microbial community.
 34. The method according to claim 28 whereinthe CSC down regulates production of polysaccharides in a microbialcommunity.
 35. A method of enhancing microbial digestion of sewage at asewage treatment plant comprising: assessing at least one of volume,flow rate, Biochemical Oxygen Demand/Chemical Oxygen Demand loading, andbacterial composition of said sewage to determine a dosage of at leastone cell signaling chemical (CSC); and adding the dosage of the at leastone cell signalling chemical to said sewage at the sewage treatmentplant or as said sewage arrives at the sewage treatment plant, whereinthe at least one CSC enhances the activity of at least one of aerobic,facultative anaerobic and anaerobic bacteria by modulating geneexpression within a microbial cell or modulating communication signalsbetween microbial cells or populations of microbial cells, and whereinthe at least one CSC does not kill said bacteria.
 36. The methodaccording to claim 35 wherein the at least one CSC is selected from thegroup consisting of N-acyl homoserine lactones (AHLs), pheromonepeptides, N-acetylated, C-amidated D-amino acid hexapeptides, D-aminoacid peptides comprising D-isoleucine and/or D-tyrosine, cyclicdipeptides, hydrophobic tryamines, lipopeptide biosurfactants, fattyacid derivatives, antimicrobial peptides and furanones.
 37. The methodaccording to claim 35 wherein the CSC is an N-acyl homoserine lactone,an N-acetylated, C-amidated D-amino acid hexapeptide or a mixturethereof.
 38. A method of managing methane gas production at a sewagetreatment plant comprising: assessing at least one of volume, flow rate,Biochemical Oxygen Demand/Chemical Oxygen Demand loading, and bacterialcomposition of sewage in a sewage treatment system to determine a dosageof at least one cell signaling chemical (CSC); and adding the dosage ofthe at least one cell signalling chemical to said sewage in the sewagetreatment system, wherein the at least one CSC enhances or inhibits theactivity of anaerobic, methane forming bacteria by modulating geneexpression within a microbial cell or modulating communication signalsbetween microbial cells or populations of microbial cells, and whereinthe at least one CSC does not kill said bacteria.
 39. A method ofenhancing digestion of sewage sludge comprising: assessing at least oneof volume, flow rate, Biochemical Oxygen Demand/Chemical Oxygen Demandloading, and bacterial composition of sewage containing said sewagesludge to determine a dosage of at least one cell signaling chemical(CSC); and adding the dosage of the at least one cell signallingchemical to said sewage, wherein the at least one CSC enhances theactivity of aerobes, facultative anaerobes, anaerobic bacteria, orcombinations thereof by modulating gene expression within a microbialcell or modulating communication signals between microbial cells orpopulations of microbial cells, and wherein the at least one CSC doesnot kill said bacteria.
 40. The method according to claim 39 wherein theat least one CSC is selected from the group consisting of N-acylhomoserine lactones, Zeatin, 6-(-(γγ-dimethylallylamino)purine,6-benzylamino-purine, kinetin, indole-3-acetic acid, and Rhodomine 123.41. A method of resuscitating dormant microbes, or microbes that are ina starvation or stationary phase in sewage comprising: assessing atleast one of volume, flow rate, Biochemical Oxygen Demand/ChemicalOxygen Demand loading, and bacterial composition of said sewage todetermine a dosage of at least one cell signaling chemical (CSC); andadding the dosage of the at least one cell signalling chemical to saidsewage, wherein the at least one CSC stimulates activity in the dormant,starved or stationary microbes by modulating gene expression within amicrobial cell or modulating communication signals between microbialcells or populations of microbial cells, and wherein the at least oneCSC does not kill said microbes.
 42. The method according to claim 41wherein the CSC is a furanone involved in cross talk with AHLs orpeptide pheromones or mixtures thereof.
 43. A method of controllingbacteria responsible for oxidation or reduction of nitrogenous compoundsin sewage comprising: assessing at least one of volume, flow rate,Biochemical Oxygen Demand/Chemical Oxygen Demand loading, and bacterialcomposition of said sewage to determine a dosage of at least one cellsignaling chemical (CSC); and adding the dosage of the at least one cellsignalling chemical (CSC) to said sewage, wherein the at least one CSCregulates the activity of ammonia producing bacteria, nitrite producingbacteria, nitrous oxide producing bacteria, nitrate producing bacteriaor denitrifying bacteria by modulating gene expression within amicrobial cell or modulating communication signals between microbialcells or populations of microbial cells, and wherein said at least oneCSC does not kill said bacteria.
 44. The method according to claim 43wherein the at least one CSC is selected from the group consisting ofhalogenated furanones, hydroxylated furanones, alkyl furanones, N-acylhomoserine lactones, peptide pheromones and mixtures thereof.
 45. Themethod according to claim 43 wherein the at least one CSC is selectedfrom the group consisting of 3-oxo-decanoyl homoserine lactone, butyrylhomoserine lactone and mixtures thereof.
 46. A method of reducingmicrobial activity in a sewerage catchment network comprising: assessingat least one of volume, flow rate, Biochemical Oxygen Demand/ChemicalOxygen Demand loading, and bacterial composition of sewage in saidsewerage catchment network to determine a dosage of at least one cellsignaling chemical (CSC); and adding the dosage of the at least one cellsignalling chemical to said sewage, wherein adding the at least one CSCto a sewerage catchment network down-regulates microbial activity ofaerobic bacteria, facultative anaerobic bacteria, anaerobic bacteria, orcombinations thereof in sewage by modulating gene expression within amicrobial cell or modulating communication signals between microbialcells or populations of microbial cells, and wherein the at least oneCSC does not kill said bacteria.